This invention was supported in part through a grant or award from the National Institute of Health. The U.S. Government, therefore, may have certain rights to this invention.
This invention generally relates to the development of vaccines against rotavirus-induced diarrheal disease and methods of using them. More specifically, the invention relates to the development of an improved parenteral immunization using live or inactivated rotavirus preparations, alone or in combination with each other or in combination with an oral vaccine or a rotavirus subunit vaccine.
Rotaviruses are the single most important pathogen causing severe diarrhea in children in both developed and developing countries. Conner et al., Current Topics in Microbiology and Immunology, in press (1992). Rotavirus infections result in over 500,000 deaths each year among children less than 2 years of age in developing countries. Institute of Medicine, xe2x80x9cProspects for immunizing against rotavirusxe2x80x9d in New Vaccine Development, Establishing Priorities. Vol. 1Diseases of Importance in the United States. (National Academy Press, Washington D.C. 1985). For children with rotavirus infections in developed countries, the case mortality rate is lower, but hospitalizations are frequent.
In the United States, despite the effectiveness and availability of oral rehydration solutions, about 11% of children with symptomatic rotavirus infections who seek medical care become moderately dehydrated and require hospitalization. Koopman et al., Am. J. Epidemiol. 119:114-123 (1984); Rodriguez et al., Pediatr. Infect, Dis. J., 6:170-176 (1987); Glass et al., J. Pediatr., 118:27-33 (1991). The Centers for Disease Control has estimated that 220,000 children are hospitalized for gastroenteritis in the United States each year and that more than half of these children have rotavirus-associated illness. Ho et al., J. Infect. Dis., 158:1112-1116 (1988); LeBaron et al., Morbid. Mortal. Weekly Rep., 39:1-14 (1990); Glass et al. (1991). A recent analysis of the effect of rotavirus infections at a large pediatric hospital in Houston, Tex., estimated that the risk for hospitalization for rotavirus gastroenteritis during childhood is 1 in 48; the extrapolated hospital bed costs alone for the United States were $352 million annually. Matson and Estes, J. Infect. Dis. 162:598-604 (1990). This estimate agrees closely with the Centers for Disease Control estimate that the annual inpatient cost of rotavirus-gastroenteritis is one billion dollars. Le Baron et al. (1990). Such data emphasize the need for a vaccine to prevent rotavirus-induced gastroenteritis during the first 2 years of life.
Several strategies have been pursued for development of a rotavirus vaccine for children. Conner et al, (1992); Kapikian et al., Adv. Exp. Med. Biol. 257:67-90 (1990). To date, most effort has been focused on the development and testing of live oral vaccines for children because these were assumed to be necessary to stimulate local mucosal antibody. Kapikian et al. (1990). Unfortunately, vaccination of young children with live oral animal (bovine, rhesus) or human (M37) vaccines or animal-human reassortant vaccines has not yet achieved sufficient take rates with good heterotypic protection in all settings. Conner et al., (1992).
The presence of pre-existing maternal antibody in children administered oral live rotavirus vaccine can interfere with the replication of the vaccine virus, and therefore, reduce the take rate of the vaccine. Cadranel et al., J. Pediatr. Gastroenterol. Nutr. 6: 525-528 (1987); Tajimra et al., Vaccine 8:71-74 (1990). Interference by maternal antibody should not be a problem with parenterally administered vaccines. Additionally, multivalent vaccines are being tested to stimulate heterotypic immunity, but achieving a balanced formulation of several live viruses has proven difficult. Perez-Schael et al., J. Clin. Microbiol. 28:553-558 (1990); Flores et al., Lancet 336:330-334 (1990); Vesikari et al., Vaccine 9:334-339 (1991); Wright et al., J. Infect. Dis. 164:271-276 (1991).
Alternative strategies using non-replicating subunit vaccines have been proposed, but to date, the ability of such vaccines to induce active protective immunity has not been demonstrated. One reason for this is that relatively few animal models are available to test the ability of a vaccine candidate to induce active protection. For example, only passive protection can be studied in the widely used neonatal mouse model of rotavirus infection because mice are only susceptible to diarrheal disease up to 14 days of age. Wolf et al., Infect. Immun. 33:565-574 (1981); Ramig, Microb. Pathog. 4:189-202 (1988).
A model of rotavirus infection in rabbits that mimics infections in children has been developed. The model is useful to monitor the development of active serum and mucosal immunity and protection from challenge against rotavirus. Conner et al., J. Virol. 62:1625-1633 (1988); Conner et al., J. Virol. 65:2563-2571 (1991); Thouless et al., Arch. Virol. 89:161-170 (1986); Hambraeus et al., Arch. Virol. 107:237-251 (1989).
Currently, measurement of protection in the rabbit model is not based on clinical illness, as diarrhea is not consistently seen in the rabbit following rotavirus inoculation due to the extremely efficient fluid absorption of the rabbit cecum. However, histopathologic changes observed over the entire length of the small intestine of infected rabbits (Gilger et al., Gastroenterology 194:A146 (1989)), changes in the amount and consistency of intestinal fluid, and the kinetics of virus shedding after infection of antibody-negative rabbits with virulent Ala virus are evidence of productive virus infection, as seen in experimental infections in other animal models and in natural infections in children where clinical diarrhea is observed. Other experiments have shown that detection of infectious virus by plaque assay and excretion of rotavirus antigen by ELISA were of comparable sensitivity. Conner et al. (1988).
The ability of anti-rotavirus IgG to mediate protection has previously been reported in passive protection studies in the suckling mouse model. Offit and Clark, J. Virol. 54:58-64 (1985); Offit and Dudzik, J. Clin. Microbiol. 27:885-888 (1989); Offit et al., J. Virol. 58:700-703 (1986); Matsui et al., J. Clin. Micobiol. 27:780-782 (1989); Sheridan et al., J. Infect Dis. 149:434-438 (1984). The ability of IgG present in the intestine to mediate protection from infection, ameliorate disease, or reduce virus excretion in children has been shown by the use of bovine milk or serum immunoglobulin from hyperimmunized cows for passive treatments in children. Barnes et al., Lancet 1:1371-1373 (1982); Losonsky et al., J. Clin. Invest. 76:2362-2367 (1985); Brussow et al., J. Clin Microbiol. 25:932-986 (1987); Hilpert et al., J. Infect. Dis. 156:158-166 (1987). Circulating anti-rotavirus antibody which mediated protection in colostrum-deprived calves has been reported following administration of high titer antibody by subcutaneous injection. Besser et al., J. Virol. 62:2238-2242 (1988a). This protection was shown to be mediated by the transfer of circulating IgG, to the intestine. Besser et al. (1988a); Besser et al., J. Virol. 62:2234-2237 (1988b).
Little information is available on the direct comparisons of live and inactivated virus as it relates to changes in immunogenicity of inactivated rotaviruses. Inactivation of bovine RIT 4237 rotavirus strain by formalin was reported to cause alterations of the virus and parenteral (intramuscular) or intragastric vaccinations with such virus failed to induce cross-protection of piglets challenged with human rotaviruses. Zissis et al., J. Infec. Dis. 148:1061-1068 (1983). Inactivation of rotavirus strain RRV by xcex2-propiolactone has been reported to cause changes in VP4 reactivity, determined by comparison of hemagglutination titers of live and inactivated virus, although passive protection of mice pups still was achieved. Offit and Dudzik (1989).
It is therefore an object of the invention to provide a vaccine against rotavirus that can be used parenterally.
It is a another object of the invention to provide a vaccine against rotavirus that uses live or inactivated rotavirus preparations, alone or in combination with each other and with or without adjuvants.
It is still another object of the invention to provide a live or inactivated vaccine which can be administered alone or in combination with a rotavirus subunit vaccine or oral rotavirus vaccine.
A feature of this invention is that the rotavirus used in the vaccine may be any cultivatable serotype 3 rotavirus or any other cultivatable serotype where a human rotavirus is represented.
Another feature of this invention is that immunization may be achieved by active immunization of the vaccine (infant, adult or animal) or through passive immunization of the infant or young animal by immunization of its mother prior to birth.
One aspect of the present invention is a live or inactivated parenteral vaccine against rotavirus infection comprising a virus classified in a rotavirus serotype which includes at least one human rotavirus.
There is provided in accordance with another aspect of the present invention a method of immunizing humans or animals with a live or inactivated parenteral vaccine against rotavirus infection comprising a virus classified in a rotavirus serotype which includes at least one human rotavirus.
Further objects, features and advantages will be apparent from the following description of the preferred embodiments of the invention.
FIG. 1. Formalin inactivation curve of SA11 rotavirus. Titers of infectious virus (PFU per milliliter) were determined by plaque assay in MA-104 cells at indicated times for untreated virus (-♦-), virus incubated at 37xc2x0 C. without addition of formalin (-▪-), and virus inactivated with formalin (-xe2x96xa1-).
The rabbit has been shown to be a useful model to examine the induction of active immunity and protection against rotavirus. The rabbit model, therefore, was used here to determine whether parenteral vaccination could induce an immune response that protected rabbits from homotypic challenge with a virulent rabbit (Ala) rotavirus. Rabbits were vaccinated with live or inactivated serotype 3 rotavirus, SA11, preparations. The virus may be used by itself as a vaccine. A virus-adjuvant formulation, however, is preferred.
Inactivated virus was prepared by treatment with formalin. Other inactivating agents, e.g., xcex2-propiolactone, or any virus inactivation technique well known in the art also may be used. Inactivation of the virus was determined by plaque assay. Live and inactivated virus preparations were diluted in phosphate-buffered saline (PBS) and combined with one of two adjuvants, either Freund""s adjuvant or aluminum phosphate. Freund""s adjuvant is a very potent immunomodulating substance. Aluminum phosphate is an FDA-approved adjuvant for use in humans, as is aluminum hydroxide. Other adjuvants such as calcium phosphate,bacille Calmette-Guerin, Corynebacterium parvum and Bordetella pertussis, are widely used in the art and may be substituted here.
Five to six month old rabbits from a rotavirus free colony were tested. All rabbits, except controls, were vaccinated intramuscularly with virus-adjuvant or PBS- adjuvant formulations. The vaccinated rabbits were vaccinated either once or twice. The amount of virus in each dose is about 1xc3x97102 to about 1xc3x97108 PFU/ml; preferably about 1xc3x97107 PFU/ml. After vaccination, all the vaccinated rabbits and controls were challenged orally with at least 1xc3x97102 PFU, preferably about 3 xc3x97105 PFU, of virulent Ala rotavirus.
For oral challenge, 1 ml of undiluted virus or virus diluted in PBS was administered from a syringe peros using a blunt-ended feeding needle as described in Conner et al. (1988).
For single vaccine dosed rabbits, challenge with virulent Ala rotavirus may be administered about at least 14 days post-vaccination (dpv), preferably about at least 21 dpv. For twice vaccine dosed rabbits, the initial dose was administered at 0 dpv, the second dose may be administered at least 14 dpv (preferably at least 21 dpv), and the oral challenge may be administered from at least 7 days (preferably about 21 days) after the second dose. Based on the rabbit model, those skilled in the art will realize that vaccination of human infants may be done in accordance with routine immunization schedules.
Intestinal lavage and serum samples were collected from all rabbits at the timepoints outlined below in the specific examples and as previously described in Conner et al. (1991). Because of the time requirements for performing the ravage procedure, a maximum of ten rabbits could be sampled on one day. Therefore, the lavage and corresponding serum samples were collected over two days (consecutive days whenever possible) for each timepoint. For data analysis, no distinction was made between the two sampling days. Fecal samples were collected 0 to 10 days post-challenge (dpc).
Antigen ELISAs were performed using a modification of the procedure described previously in Conner et al. (1988) to measure rotavirus excretion. Briefly, the modifications were as follows: Polyvinylchloride plates were coated with 100 xcexcl of hyperimmune guinea pig anti-Ala serum overnight at room temperature, and the wells were blocked with 200 xcexcl of 5% skim milk in PBS for 2 hours at 37xc2x0 C. The reagent volume for each subsequent step was 100 xcexcl. The conjugate was diluted in 0.5% skim milk in PBS. A sample was considered positive if the A414 value of the mean duplicate wells was greater than 0.1 and this absorbance value was greater than or equal to two standard deviations above the absorbance value of the negative diluent control. Serial dilutions of Ala stock virus also were included on each plate as a positive control.
ELISAs to measure total (IgA, IgM, IgG) anti-rotavirus antibody in serum and intestinal samples were performed as previously described in Conner et al. (1991) to measure antibody responses. The ELISAs to measure IgA concentration and anti-rotavirus IgA antibody in serum and intestinal samples were performed using a modification of the procedures previously described in Conner et al., (1991). Specifically, the assays were modified by use of (i) a monoclonal antibody specific for rabbit IgA (Cole, Monoclonal Antibody News 7:23-24 (1989)) (supplied by Carol Cole of Naval Research, Bethesda, Md.) conjugated to biotin (Guesdon et al., J. Histo. Cyto. 27:1131-1139 (1979)), (ii) an avidin-horseradish peroxidase (HRP) conjugate (Vector Laboratories, Inc., Burlingame, Calif.), and (iii) a TMB (3,3xe2x80x2,5,5xe2x80x2-tetramethylbenzidine) substrate (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.). Total IgA concentrations were determined for pre- and post-challenge intestinal lavage samples. Because the IgA concentration in sequential samples for individual rabbits only varied from 1- to 4.4-fold, IgA anti-rotavirus titers were not normalized, as described previously (Conner et al. 1991)).
ELISAs to measure Ikg anti-rotavirus antibody in intestinal samples were performed identically to the total antibody ELISA (Conner et al. (1991)), except. HRP-conjugated goat anti-rabbit IgG (Hyclone Laboratories, Inc., Logan, Utah) was used as the conjugate.
All antibody titers were compared using the Wilcoxon rank sum two-sample test for unpaired samples. Because of the small number of rabbits in each vaccine group, statistical comparisons for antibody titers and days of virus excretion were made only between live and inactivated virus (in either aluminum phosphate or Freund""s adjuvant) or between virus (live and inactivated)-Freund""s adjuvant and virus (live and inactivated)-aluminum phosphate. The comparison of the means of days of virus excretion was performed using the Student""s t test. Protection from live Ala challenge was analyzed using the Fishers exact test, two-tailed.
In the instant experiments, rabbits were protected from challenge with Ala virus after two, but not one, vaccinations with live or inactivated SA11 virus in either Freund""s adjuvant or aluminum phosphate. Protection in these experiments was evaluated by examining virus shedding after challenge. None of the rabbits vaccinated twice excreted detectable virus, as detected by ELISA, whereas all control rabbits excreted virus (mean duration of shedding, 5 days). Protection from virus infection (virus excretion) following parenteral vaccination of rabbits was associated with the presence of anti-rotavirus IgG in the intestine but not anti-rotavirus IgA, as IgA was not detected in the intestine of virus-vaccinated rabbits until after the oral challenge. These results suggest that intestinal anti-rotavirus IgG antibody may mediate protection. The prior art antibody studies identified in the background of the invention support a hypothesis that protection of the rabbits was mediated by the anti-rotavirus IgG. However, the prior art studies differ significantly from the instant results in that the IgG in instant experiments was induced by active immunity stimulated by parenteral inoculation and not by passive lactogenic immunity or oral administration of IgG.
Very slight differences in reactivity to monoclonal antibodies to rotavirus surface antigens VP7 or VP4 or polyclonal antibodies to whole virus were observed by ELISA when the inactivated and live rotavirus preparations were compared prior to mixing with the adjuvants. Immune responses and protection also were compared using the two adjuvants, Freund""s adjuvant and aluminum phosphate. Although the serum and intestinal antibody titers induced by live and inactivated vaccines in the two adjuvants did not appear to vary, it was not possible to statistically compare the antibody titers due to the small number of rabbits in each group. Titers induced by the two adjuvants, however, were compared by grouping the rabbits by type of adjuvant. Following two vaccinations, significantly higher serum anti-rotavirus titers were induced in rabbits vaccinated with Freund""s adjuvant as compared to rabbits vaccinated with aluminum phosphate adjuvant, although protection was observed in both groups. However, these two adjuvants did not induce significant differences in IgG or total anti-rotavirus titers in the intestine, which would account for the equivalent levels of protection induced by both vaccine-adjuvant formulations.
A statistically significant difference in serum titers induced by the two adjuvants remained following challenge. This was not surprising, since little or no increase in serum titer was observed following challenge. Since these rabbits were protected from detectable virus infection, it is likely that no or minimal viral replication occurred in the intestine. The input dose of challenge virus or the limited virus replication might be sufficient to induce or boost an intestinal but not a serologic response. Intestinal antibody induction (IgA) was observed in all but one of the rabbits and boosting of IgG and total Ig was observed in three of the rabbits.
All but one of the virus-vaccinated rabbits had detectable IgA responses following oral challenge with live virus. These results indicate that a combined parenteral-oral or oral-parenteral vaccination regimen also could be used to induce intestinal IgG and IgA immunity.
The present invention is not restricted to the use of simian rotavirus SA11 clone 3 which is herein described and used for exemplification purposes only. The invention applies to the use of any cultivatable serotype 3 rotavirus or any other cultivatable serotype where a human rotavirus is represented. Serotype, as it is used herein, means a classification of viruses by a specific neutralizing antibody/antigen reaction.